Studies on methods of extracting natural mixture of conjugated estrogens from pregnant mares' urine (PMU) have been reported since 1930s. Because of its definite therapeutic effect and safety reliability, mixture of natural conjugated estrogens is used in clinic for treating and preventing female physiological or artificial climacteric period syndrome occurred after menopause. The mixture can also be used to treat and prevent osteoporosis, the effect of which has been widely recognized. The extracting methods have been improved continuously during the past 70 years, especially for the recent years.
The early reports on methods of extracting conjugated estrogens mixture include U.S. Pat. No. 2,429,398, U.S. Pat. No. 2,519,516, U.S. Pat. No. 2,696,265, U.S. Pat. No. 2,711,988, U.S. Pat. No. 2,834,712, etc., where organic solvents were mainly applied for the purpose of extraction. After 1950s, active carbon, ion exchange resin, and reverse phase silica gel, etc. was used in methods for extracting a conjugated estrogens mixture from PMU. However, the adsorptive capacities achieved by these methods were low, which is not suitable for large-scale production.
In late 1990s, the application of new macroporous adsorption resin in obtaining mixture of conjugated estrogens from PMU showed great advantages. U.S. Pat. No. 5,723,454 (Ivan Ban., et al.) describes a method of obtaining a extract containing mixture of natural conjugated estrogens from PMU by using non-ion semipolar macroporous cross-linked polyacrylate resin; CN 1381240A (Song Walapa, et al.) describes a method of extracting conjugated estrogens mixture from PMU by polystyrene resin; CN1526721A (Zhou tao, et al.) describes an extracting method of obtaining mixture of conjugated estrogens, wherein polar adsorption resin containing sulfoxide group was used to extract the conjugated estrogens mixture from PMU, followed by eluting.
Generally, conventional method for extracting conjugated estrogens mixture with macroporous adsorption resin comprises the following steps: first, pretreating raw PMU; second, adsorbing conjugated estrogens mixture from PMU with a macroporous adsorption resin; third, washing the macroporous adsorption resin laden with the mixture of conjugated estrogens by an alkaline/neutral aqueous solution; and eluting the washed macroporous adsorption resin to obtain the conjugated estrogens mixture. Although these methods can obtain quickly and conveniently the enrichment component of natural conjugated estrogens mixture from PMU, it still could not solve the problems described below.
The contents of the conjugated estrogens mixture in PMU are quite different in diverse pregnancy periods. Urines of a pregnant mare in its 3-9 months' pregnancies (effective utilization period of PMU) were collected respectively and the contents of conjugated estrogens mixtures were tested. The contents of conjugated estrogens mixture of PMU in diverse pregnancy periods ranged from 30-300 mg/L. Within the best collecting period (5-7 months' pregnancies), the contents of conjugated estrogens mixture of PMU are generally above 100 mg/L, and even more.
Nevertheless, when conventional methods were used to treat PMU with relatively high contents of conjugated estrogens mixture, the adsorptive ratio of resin to PMU is rather low. Especially when the contents of the conjugated estrogens mixture is above 100 mg/L in PMU, the adsorptive ratio of resin to PMU will generally be less than 1:30. For large-scale production, this adsorptive ratio is difficult to satisfy practical requirement.
In addition, when conventional macroporous resins were used to adsorb PMU, impurities in PMU will also be adsorbed. Sometimes, the impurities even have preference to be adsorbed over the conjugated estrogens mixture due to the non-specific adsorption of the macroporous resin. Therefore, in order to improve adsorptive ratio, the raw materials used in conventional methods need to be pretreated, for instance, by adjusting PMU to a pH of above 10 with alkaline substances or by depleting cresol from PMU. All these procedures make the methods more difficult to operate, and fail to significantly improve the production efficiency.
Moreover, the resins used in conventional methods are relatively expensive, making no much difference in cost when compared to conventional solvent extracting processes.
Therefore, it is advantageous to improve the adsorptive ability of resin to conjugated estrogens mixture in PMU and to lower the cost. The present invention intends to solve the above-mentioned problems and therefore provide a method suitable for the large-scale manufacture of raw materials for pharmaceuticals production.